Ion exchange proteins, that perform tightly-coupled one-for-one exchange of ions, such as Na+ for H+ or Cl- for HCO3-, are important in cell ion, pH, and volume regulation, as well as for the flow of ions across epithelia and for body acid-base regulation. Although the function of these proteins is critical for normal operation of organs such as the kidney, stomach, intestine, heart, and brain, and dysfunction leads to disease, their molecular structural mechanism is unknown. The human red blood cell AE1 anion exchange protein will be used as a model to examine this question. The ion exchange mechanism involves alternation between two conformations, Ei with the anion binding site facing the cytosol, and Eo with it facing the external medium. The shift from Ei - Eo or vice-versa is greatly facilitated when Cl- or HCO3- is bound to the transport site. Because of this, the transport cycle involves flow of one anion inward, followed by another anion outward. In order to discover what conformational changes in AE 1 take place during the transport cycle, methods will be developed for using substrates or inhibitors to recruit AE1 into particular forms (e.g. Ei or Eo). The question of whether halides (e.g. C1-) and oxyanions (e.g. HCO3-) bind to the same or different sites will also be examined, by using chemical probes and nuclear magnetic resonance (NMR). Next, fluorescence (FRET) and novel luminescence (LRET) energy transfer methods will be used, along with mutant AE1's containing single SH sites, to determine distances between various parts of the AE1 dimer, as well as the location of inhibitor binding sites that are known to be affected by the Ei - Eo change or by substrate binding. Finally, changes in these distances caused by transport-related changes in AE1 conformation will be used to determine which parts of AE1 move and in which direction when substrates bind and when the Ei - Eo conversion takes place. Together with other structural information, these measurements of dynamic conformational changes during transport will provide insights into the molecular mechanism of this and other exchange transporters, leading to a better understanding of how they function normally, how function is perturbed in disease, and possibly how function might be restored.